scholarly journals Sequencing Introduced False Positive Rare Taxa Lead to Distorted Microbial Community Diversity, Assembly, and Interaction Interpretation in Amplicon Studies

2021 ◽  
Author(s):  
Yangyang JIA ◽  
Shengguo Zhao ◽  
Wenjie Guo ◽  
Ling Peng ◽  
Fang Zhao ◽  
...  
Keyword(s):  
Community Assembly ◽  
False Positive ◽  
False Negative ◽  
Alpha Diversity ◽  
Amplicon Sequencing ◽  
False Positives ◽  
Community Diversity ◽  
Rare Taxa ◽  
Sequencing Platform ◽  
Sequencing Platforms

Abstract BackgroundDue to the inherent scarcity of the microbial rare taxa, it is difficult to distinguish between bona fide rare taxa and potential false positives in metabarcoding amplicon sequencing studies. Although recently developed quality control and clustering or denoising algorithms could remove sequencing errors or non-biological artifact reads, no algorithm could remove high quality reads from sample-wise cross contaminations introduced by index misassignment. ResultsWe thoroughly evaluated the rate of index misassignment of the mostly used NovaSeq 6000 and DNBSEQ-G400 sequencing platforms using both commercial and customized mock communities, and observed significant higher (5.68% vs 0.08%) fraction of potential false positive reads for NovaSeq 6000 compared to DNBSEQ-G400 in. Significant batch effects could be caused by the randomly detected false positive or false negative rare taxa. These false detections introduced by index misassignment could also lead to inflated alpha diversity for relatively simple samples while underestimated alpha diversity for complex samples. Further test using a set of cow rumen samples reported differential rare taxa by different sequencing platforms. Correlation test of the rare taxa detected by each sequencing platform demonstrated that the Novaseq 6000 platform detected rare taxa had a much lower possibility to be correlated with the physiochemical properties of rumen fluid. Community assembly mechanism and microbial network association analysis indicated that false positive or negative rare taxa detection could lead to distorted community assembly process and identification of even fake keystone species of the community, one of which was confirmed negative by PCR cloning and following Sanger sequencing. ConclusionsMetabarcoding amplicon sequencing process may introduce scarce but not neglectable false positives. We highly suggest proper positive and negative controls and technical replicate settings, and proper sequencing platform selection in future amplicon studies, especially when the microbial rare biosphere should be focused.

Evaluation of Positive T- and B-Cell Gene Rearrangement Studies Among Patients Without a Definitive Diagnosis by Other Assays

2019 ◽  
Vol 152 (Supplement_1) ◽  
pp. S35-S36
Author(s):  
Hadrian Mendoza ◽  
Christopher Tormey ◽  
Alexa Siddon
Keyword(s):  
T Cell ◽  
False Positive ◽  
Gene Rearrangement ◽  
Hematologic Malignancy ◽  
False Negative ◽  
False Positives ◽  
False Negatives ◽  
True Negative ◽  
Flow Cytometric ◽  
Pathology Reports

Abstract In the evaluation of bone marrow (BM) and peripheral blood (PB) for hematologic malignancy, positive immunoglobulin heavy chain (IG) or T-cell receptor (TCR) gene rearrangement results may be detected despite unrevealing results from morphologic, flow cytometric, immunohistochemical (IHC), and/or cytogenetic studies. The significance of positive rearrangement studies in the context of otherwise normal ancillary findings is unknown, and as such, we hypothesized that gene rearrangement studies may be predictive of an emerging B- or T-cell clone in the absence of other abnormal laboratory tests. Data from all patients who underwent IG or TCR gene rearrangement testing at the authors’ affiliated VA hospital between January 1, 2013, and July 6, 2018, were extracted from the electronic medical record. Date of testing; specimen source; and morphologic, flow cytometric, IHC, and cytogenetic characterization of the tissue source were recorded from pathology reports. Gene rearrangement results were categorized as true positive, false positive, false negative, or true negative. Lastly, patient records were reviewed for subsequent diagnosis of hematologic malignancy in patients with positive gene rearrangement results with negative ancillary testing. A total of 136 patients, who had 203 gene rearrangement studies (50 PB and 153 BM), were analyzed. In TCR studies, there were 2 false positives and 1 false negative in 47 PB assays, as well as 7 false positives and 1 false negative in 54 BM assays. Regarding IG studies, 3 false positives and 12 false negatives in 99 BM studies were identified. Sensitivity and specificity, respectively, were calculated for PB TCR studies (94% and 93%), BM IG studies (71% and 95%), and BM TCR studies (92% and 83%). Analysis of PB IG gene rearrangement studies was not performed due to the small number of tests (3; all true negative). None of the 12 patients with false-positive IG/TCR gene rearrangement studies later developed a lymphoproliferative disorder, although 2 patients were later diagnosed with acute myeloid leukemia. Of the 14 false negatives, 10 (71%) were related to a diagnosis of plasma cell neoplasms. Results from the present study suggest that positive IG/TCR gene rearrangement studies are not predictive of lymphoproliferative disorders in the context of otherwise negative BM or PB findings. As such, when faced with equivocal pathology reports, clinicians can be practically advised that isolated positive IG/TCR gene rearrangement results may not indicate the need for closer surveillance.

scholarly journals Comparison of PCR versus PCR-Free DNA Library Preparation for Characterising the Human Faecal Virome

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2093
Author(s):  
Shen-Yuan Hsieh ◽  
Mohammad A. Tariq ◽  
Andrea Telatin ◽  
Rebecca Ansorge ◽  
Evelien M. Adriaenssens ◽  
...  
Keyword(s):  
Alpha Diversity ◽  
Diversity Indices ◽  
P Value ◽  
Library Preparation ◽  
High Genetic Diversity ◽  
Sequencing Platform ◽  
Viral Genomes ◽  
Healthy Donors ◽  
Faecal Samples ◽  
Sequencing Platforms

The human intestinal microbiota is abundant in viruses, comprising mainly bacteriophages, occasionally outnumbering bacteria 10:1 and is termed the virome. Due to their high genetic diversity and the lack of suitable tools and reference databases, the virome remains poorly characterised and is often referred to as “viral dark matter”. However, the choice of sequencing platforms, read lengths and library preparation make study design challenging with respect to the virome. Here we have compared the use of PCR and PCR-free methods for sequence-library construction on the Illumina sequencing platform for characterising the human faecal virome. Viral DNA was extracted from faecal samples of three healthy donors and sequenced. Our analysis shows that most variation was reflecting the individually specific faecal virome. However, we observed differences between PCR and PCR-free library preparation that affected the recovery of low-abundance viral genomes. Using three faecal samples in this study, the PCR library preparation samples led to a loss of lower-abundance vOTUs evident in their PCR-free pairs (vOTUs 128, 6202 and 8364) and decreased the alpha-diversity indices (Chao1 p-value = 0.045 and Simpson p-value = 0.044). Thus, differences between PCR and PCR-free methods are important to consider when investigating “rare” members of the gut virome, with these biases likely negligible when investigating moderately and highly abundant viruses.

scholarly journals Automated detection of cribriform growth patterns in prostate histology images

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Pierre Ambrosini ◽  
Eva Hollemans ◽  
Charlotte F. Kweldam ◽  
Geert J. L. H. van Leenders ◽  
Sjoerd Stallinga ◽  
...  
Keyword(s):  
Deep Learning ◽  
False Positive ◽  
Clinical Decision Making ◽  
False Negative ◽  
Area Under The Curve ◽  
High Sensitivity ◽  
False Positives ◽  
Growth Patterns ◽  
Learning Method ◽  
Method Performance

Abstract Cribriform growth patterns in prostate carcinoma are associated with poor prognosis. We aimed to introduce a deep learning method to detect such patterns automatically. To do so, convolutional neural network was trained to detect cribriform growth patterns on 128 prostate needle biopsies. Ensemble learning taking into account other tumor growth patterns during training was used to cope with heterogeneous and limited tumor tissue occurrences. ROC and FROC analyses were applied to assess network performance regarding detection of biopsies harboring cribriform growth pattern. The ROC analysis yielded a mean area under the curve up to 0.81. FROC analysis demonstrated a sensitivity of 0.9 for regions larger than $${0.0150}\,\hbox {mm}^{2}$$ 0.0150 mm 2 with on average 7.5 false positives. To benchmark method performance for intra-observer annotation variability, false positive and negative detections were re-evaluated by the pathologists. Pathologists considered 9% of the false positive regions as cribriform, and 11% as possibly cribriform; 44% of the false negative regions were not annotated as cribriform. As a final experiment, the network was also applied on a dataset of 60 biopsy regions annotated by 23 pathologists. With the cut-off reaching highest sensitivity, all images annotated as cribriform by at least 7/23 of the pathologists, were all detected as cribriform by the network and 9/60 of the images were detected as cribriform whereas no pathologist labelled them as such. In conclusion, the proposed deep learning method has high sensitivity for detecting cribriform growth patterns at the expense of a limited number of false positives. It can detect cribriform regions that are labelled as such by at least a minority of pathologists. Therefore, it could assist clinical decision making by suggesting suspicious regions.

scholarly journals Assessing the Clinical Impact of Prostate-Specific Antigen Assay Variability and Nonequimolarity: A Simulation Study Based on the Population of the United Kingdom

2004 ◽  
Vol 50 (6) ◽  
pp. 1012-1016 ◽  
Author(s):  
Andrew W Roddam ◽  
Christopher P Price ◽  
Naomi E Allen ◽  
Anthony Milford Ward ◽  
Keyword(s):  
United Kingdom ◽  
Prostate Specific Antigen ◽  
False Positive ◽  
False Negative ◽  
Specific Antigen ◽  
False Positives ◽  
False Negatives ◽  
Worst Case ◽  
The United Kingdom ◽  
Analytical Imprecision

Abstract Background: Prostate-specific antigen (PSA) is the most widely used serum biomarker to differentiate between malignant and benign prostate disease. Assays that measure PSA can be biased and/or nonequimolar and hence report significantly different PSA values for samples with the same nominal amount. This report investigates the effects of biased and nonequimolar assays on the decision to recommend a patient for a prostate biopsy based on age-specific PSA values. Methods: A simulation model, calibrated to the distribution of PSA values in the United Kingdom, was developed to estimate the effects of bias, nonequimolarity, and analytical imprecision in terms of the rates of men who are recommended to have a biopsy on the basis of their assay-reported PSA values when their true PSA values are below the threshold (false positives) or vice versa (false negatives). Results: False recommendation rates for a calibrated equimolar assay are 0.5–0.9% for analytical imprecision between 5% and 10%. Positive bias leads to significant increases in false positives and significant decreases in false negatives, whereas negative bias has the opposite effect. False-positive rates for nonequimolar assays increase from 0.5% to 13% in the worst-case scenario, whereas false-negative rates are almost always 0%. Conclusions: Biased and nonequimolar assays can have major detrimental effects on both false-negative and false-positive rates for recommending biopsy. PSA assays should therefore be calibrated to the International Standards and be unbiased and equimolar in response to minimize the likelihood of incorrect clinical decisions, which are potentially detrimental for both patient and healthcare provider.

scholarly journals CORP: Minimizing the chances of false positives and false negatives

2017 ◽  
Vol 122 (1) ◽  
pp. 91-95 ◽  
Author(s):  
Douglas Curran-Everett
Keyword(s):  
Hypothesis Testing ◽  
Experimental Technique ◽  
False Positive ◽  
Scientific Discovery ◽  
False Negative ◽  
False Positives ◽  
Experimental Result ◽  
False Negatives ◽  
Scientific Equipment ◽  
Recent Initiative

Statistics is essential to the process of scientific discovery. An inescapable tenet of statistics, however, is the notion of uncertainty which has reared its head within the arena of reproducibility of research. The Journal of Applied Physiology’s recent initiative, “Cores of Reproducibility in Physiology,” is designed to improve the reproducibility of research: each article is designed to elucidate the principles and nuances of using some piece of scientific equipment or some experimental technique so that other researchers can obtain reproducible results. But other researchers can use some piece of equipment or some technique with expert skill and still fail to replicate an experimental result if they neglect to consider the fundamental concepts of statistics of hypothesis testing and estimation and their inescapable connection to the reproducibility of research. If we want to improve the reproducibility of our research, then we want to minimize the chance that we get a false positive and—at the same time—we want to minimize the chance that we get a false negative. In this review I outline strategies to accomplish each of these things. These strategies are related intimately to fundamental concepts of statistics and the inherent uncertainty embedded in them.

scholarly journals Fungal alpha-diversity drives the stochasticity of bacterial and fungal community assembly in soil aggregates in the apple orchard

2020 ◽  
Author(s):  
Wei Zheng ◽  
Zhiyuan Zhao ◽  
Fenglian Lv ◽  
Yanan Yin ◽  
Zhaohui Wang ◽  
...  
Keyword(s):  
Bacterial Community ◽  
Fungal Community ◽  
Community Assembly ◽  
Fungal Diversity ◽  
Soil Aggregates ◽  
Alpha Diversity ◽  
Apple Orchard ◽  
Community Diversity ◽  
Agricultural Practice ◽  
Bacteria And Fungi

Abstract Background In soil ecosystems, bacteria and fungi always co-exist in the same niche and interact with each other, especially in different sized soil aggregates. The bacterial and fungal community assembly process and bacteria-fungi interactions in soil aggregates, which is important for bacterial and fungal community diversity and composition, is still unclear.Methods We examined bacterial and fungal community assembly in soil macroaggregate (> 0.25 mm), microaggregate (0.053–0.25 mm) and smaller microaggregate (silt + clay, < 0.053 mm) in an apple orchard. The microbial community assembly processes were analyzed by normalized stochasticity ratio index (NST).Results Bacterial community diversity, composition and assembly were more affected by agricultural practice and aggregate than fungal community. Bacterial community assembly was more stochastic in silt + clay than in macroaggregate, and was more stochastic (NST > 50%) than fungal community in soil aggregates. Meanwhile, bacterial NST was negatively correlated with fungal diversity, and fungal NST was positively correlated with fungal diversity. Co-occurrence network suggested that the bacteria and fungi were less strongly interacting in the network of silt + clay, compared to macroaggregate. The results indicated that fungi impact on the bacterial community assembly in soil aggregate, and the stochasticity of bacterial community assembly was increased with the decrease of interaction between bacteria and fungi in soil aggregates.Conclusions This study enhances our understanding of the mechanism of bacterial and fungal community assembly and co-exists pattern of bacteria and fungi in soil aggregates.

scholarly journals Predictors of dementia misclassification when using brief cognitive assessments

2018 ◽  
Vol 9 (2) ◽  
pp. 109-117 ◽  
Author(s):  
Janice M. Ranson ◽  
Elżbieta Kuźma ◽  
William Hamilton ◽  
Graciela Muniz-Terrera ◽  
Kenneth M. Langa ◽  
...  
Keyword(s):  
Nursing Home ◽  
False Positive ◽  
Polynomial Regression ◽  
False Negative ◽  
False Positives ◽  
Cognitive Assessments ◽  
False Negatives ◽  
Cut Point ◽  
Home Residency ◽  
Poor Memory

BackgroundBrief cognitive assessments can result in false-positive and false-negative dementia misclassification. We aimed to identify predictors of misclassification by 3 brief cognitive assessments; the Mini-Mental State Examination (MMSE), Memory Impairment Screen (MIS) and animal naming (AN).MethodsParticipants were 824 older adults in the population-based US Aging, Demographics and Memory Study with adjudicated dementia diagnosis (DSM-III-R and DSM-IV criteria) as the reference standard. Predictors of false-negative, false-positive and overall misclassification by the MMSE (cut-point <24), MIS (cut-point <5) and AN (cut-point <9) were analysed separately in multivariate bootstrapped fractional polynomial regression models. Twenty-two candidate predictors included sociodemographics, dementia risk factors and potential sources of test bias.ResultsMisclassification by at least one assessment occurred in 301 (35.7%) participants, whereas only 14 (1.7%) were misclassified by all 3 assessments. There were different patterns of predictors for misclassification by each assessment. Years of education predicts higher false-negatives (odds ratio [OR] 1.23, 95% confidence interval [95% CI] 1.07–1.40) and lower false-positives (OR 0.77, 95% CI 0.70–0.83) by the MMSE. Nursing home residency predicts lower false-negatives (OR 0.15, 95% CI 0.03–0.63) and higher false-positives (OR 4.85, 95% CI 1.27–18.45) by AN. Across the assessments, false-negatives were most consistently predicted by absence of informant-rated poor memory. False-positives were most consistently predicted by age, nursing home residency and non-Caucasian ethnicity (all p < 0.05 in at least 2 models). The only consistent predictor of overall misclassification across all assessments was absence of informant-rated poor memory.ConclusionsDementia is often misclassified when using brief cognitive assessments, largely due to test specific biases.

Drug screening in urine by cloned enzyme donor immunoassay (CEDIA) and kinetic interaction of microparticles in solution (KIMS): a comparative study

2006 ◽  
Vol 44 (4) ◽  
Author(s):  
Lutz Schwettmann ◽  
Wolf-Rüdiger Külpmann ◽  
Christian Vidal
Keyword(s):  
Gas Chromatography ◽  
Drug Screening ◽  
False Positive ◽  
False Negative ◽  
False Positives ◽  
Gas Chromatography Mass Spectrometry ◽  
Electron Capture Detection ◽  
False Negatives ◽  
Negative Results ◽  
Kinetic Interaction

AbstractTwo commercially available drug-screening assays were evaluated: the Roche kinetic interaction of microparticles in solution (KIMS) assay and the Microgenics cloned enzyme donor immunoassay (CEDIA). Urine samples from known drug-abuse patients were analyzed for amphetamines, barbiturates, benzodiazepines, benzoylecgonine, cannabinoids, LSD, methadone and opiates. Samples with discordant findings for the two assays were analyzed by gas chromatography/mass spectrometry (GC/MS) or gas chromatography/electron capture detection (GC/ECD). Amphetamines showed 96.0% concordant results, with two false positive findings by CEDIA, three by KIMS and a further two false negatives by KIMS. Barbiturates showed 99.4% concordant results, with one false negative by KIMS. Benzodiazepines showed 97.4% concordant results, with two false negatives by KIMS (cutoff 100μg/L, CEDIA cutoff 300 μg/L). Benzoylecgonine showed 17.8% concordant positive and 82.2% concordant negative results and no false finding by either assay. Cannabinoids showed 99.3% concordant results, with one sample negative by KIMS at a cutoff of 50μg/L and positive by CEDIA (cutoff 25μg/L). For LSD, 6.7% of findings were not in agreement. Methadone showed 97.5% concordant results, with two false positives by CEDIA, and one false positive and one false negative by KIMS. Opiates showed 96.9% concordant results, with no false KIMS results, but four false positives by CEDIA. The results indicate that the agreement of the CEDIA and KIMS results for the eight drugs is rather good (93.3–100%).

scholarly journals Does it matter for the radiologists’ performance whether they read short or long batches in organized mammographic screening?

2021 ◽  
Author(s):  
Heinrich A. Backmann ◽  
Marthe Larsen ◽  
Anders S. Danielsen ◽  
Solveig Hofvind
Keyword(s):  
False Positive ◽  
False Negative ◽  
Population Level ◽  
Time Of Day ◽  
False Positives ◽  
Mediation Effect ◽  
False Negatives ◽  
Negatively Associated ◽  
The Individual ◽  
Image Position

Abstract Objective To analyze the association between radiologists’ performance and image position within a batch in screen reading of mammograms in Norway. Method We described true and false positives and true and false negatives by groups of image positions and batch sizes for 2,937,312 screen readings performed from 2012 to 2018. Mixed-effects models were used to obtain adjusted proportions of true and false positive, true and false negative, sensitivity, and specificity for different image positions. We adjusted for time of day and weekday and included the individual variation between the radiologists as random effects. Time spent reading was included in an additional model to explore a possible mediation effect. Result True and false positives were negatively associated with image position within the batch, while the rates of true and false negatives were positively associated. In the adjusted analyses, the rate of true positives was 4.0 per 1000 (95% CI: 3.8–4.2) readings for image position 10 and 3.9 (95% CI: 3.7–4.1) for image position 60. The rate of true negatives was 94.4% (95% CI: 94.0–94.8) for image position 10 and 94.8% (95% CI: 94.4–95.2) for image position 60. Per 1000 readings, the rate of false negative was 0.60 (95% CI: 0.53–0.67) for image position 10 and 0.62 (95% CI: 0.55–0.69) for image position 60. Conclusion There was a decrease in the radiologists’ sensitivity throughout the batch, and although this effect was small, our results may be clinically relevant at a population level or when multiplying the differences with the number of screen readings for the individual radiologists. Key Points • True and false positive reading scores were negatively associated with image position within a batch. • A decreasing trend of positive scores indicated a beneficial effect of a certain number of screen readings within a batch. • False negative scores increased throughout the batch but the association was not statistically significant.

scholarly journals Clinical performance of Non-invasive prenatal testing for sex chromosomal aneuploidies in Northeast China

2020 ◽  
Author(s):  
Lu Wang ◽  
Rulin Dai ◽  
Qingyang Shi ◽  
Yuting Jiang ◽  
Hongguo Zhang ◽  
...  
Keyword(s):  
False Positive ◽  
Northeast China ◽  
Sex Chromosome ◽  
Clinical Performance ◽  
False Positive Rate ◽  
False Negative ◽  
Prenatal Testing ◽  
Potential Method ◽  
Sequencing Platform ◽  
Non Invasive

Abstract Background: Along with the discovery of cell-free DNA (cfDNA) and the invention of next-generation sequencing (NGS), non-invasive prenatal testing (NIPT) had appeared and been applied for detecting common aneuploidies such as trisomy 21, 18, and 13, with low false-negative and false-positive rates. Recently, it had also been used for sex chromosome aneuploidies (SCAs). To assess the clinical utility of NIPT for SCAs in Northeast China, we collected NIPT data from BGI 500 sequencing platform in the Center for Reproductive Medicine, Center for Prenatal Diagnosis of the First Hospital of Jilin University, and calculate the positive predictive value (PPV) and false positive rate (FPR). Results: A cohort of 14936 samples were analyzed by NIPT, and revealed 70 cases with SCAs high-risk, among them, 40 women agreed to undergo amniocentesis, but as many as 30 ones refused further diagnose. Based on verified fetal karyotype, 30.0% (12/40) were confirmed to be a true positive. Unluckily, the PPV for monosomy X performed 0%. Besides, positive 47,XXX were 46.67% (7/15), 40.00% (2/5) were positive for 47, XYY, and 42.86% (3/7) were positive for 47, XXY.Conclusions: In conclusion, our present results confirmed that NIPT sequenced by BGI 500 demonstrated lowest PPVs for 45,X, but the more accurate prediction for other SCAs, it is still a potential method for SCAs screening. Henceforth, we should focus on how to improve the test utility and provide better services for pregnant women in need.

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